There are up to 7 species of Ureaplasma, The two species associated with human infection are Ureaplasma parvum and Ureaplasma urealyticum. All species within the genus Ureaplasma, family Mycoplasmataceae. They are prokaryotes devoid of a cell wall and hence insensitive to penicillin and gram stain. They are small (0.1-0.85 um) and best visualized in broth culture by dark-field or phase-contrast microscopy, but its pleomorphic nature makes it difficult to identify in medium. Thus, organisms typical colonies are recognized on solid medium (7-30 um) and is the sine qua non for identification. (Taylor-Robinson and Gourley, 1984)
Ureaplasma need urea for growth even in highly complex media and produce the enzyme urease which allows the organism to metabolize urea. (Pollack, 1986) They do not synthesize folic acid and as such are not susceptible to sulfonamides or trimethoprim. Ureaplasma produce hemolysin. (Furness, 1973; Shepar and Masover, 1979) Ureaplasma appear to attach to a variety of host cells via unique mechanisms and then invade the host cell. (Busolo et al., 1984; masover et al., 1977; Robertson et al., 1991; Saada et al., 1991; Shepard and Masover, 1979; Torres-Morquecho et al., 2010) This has been associated with cell apoptosis (Li et al., 2002) and increased inflammatory cytokines. Several have reported that Hela (McGarrity and Kotani, 1986; Smith et al., 1994) or A549 (Torres-Morquecho et al., 2010) cells can be used to study this attachment.
Serologic and genomic relationships among the established and unspeciated Ureaplasma species and serovars isolated from various hosts can be summarized as follows. (human) is separated into two genomic clusters (parvum and urealyticum). Ureaplasma diversum (bovine) has three serologic clusters that identify all U. diversum strains. The nonhuman primate strains form four serologic groups, and each serogroup is composed of strains isolated from primates belonging to one of four distinct zoologic primate families. The ovine-caprine strains have two serologic clusters. Canine strains form four serologic clusters but serovars 1 and 2 are closely related by DNA homology. Avian strains belong to one serogroup with two genomic clusters. (Barile M F, Pediatr Infect Dis 1986 5(6Suppl):5296-9).
U. urealyticum and U. parvum have at least 14 serotypes defined by serologic and biologic characteristics among its numerous strains. These serotypes have recently been subdivided into two biovar: U. urealyticum or group 2 (serotypes 2, 4, 5, 7, 8, 9, 10, 11, 12, 13); U. parvum or group 1 (serotypes 1, 3, 6, 14). (Robertson et al., 2001) The genome size of the various strains appears to vary widely and corresponds to the two serovar clusters. The genome size of cluster group 1 is about 760 kb, while group 2 ranges from 880 to 1,140 kb. (Robertson et al., 1990) Serovar identification can be accomplished by serology (Roberson and Stemke, 1982), immunofluorescence (Roberson and Stemke, 1982), and ELISA (Brown et al., 1981; Horotzitz et al., 1995). The latter is least labor intensive and has been reproduced (Turunen et al., 1982; Wiley and Quinn, 1984). It may be difficult to detect all serovar because of variable growth rates (Stemke and Robertson, 1985), and multiple serovar per specimen (Quinn, 1986).
The most sensitive method of isolating Ureaplasma consists of specimen inoculation into liquid medium and subculture to agar. (Robertson, 1978; Taylor-Robinson et al., 1967; Taylor-Robinson and Gourley, 1984; Taylor-Robinson, 1989) Colonies sometimes fail to develop when a specimen is plated directly on agar. In liquid medium, organisms are detected by their urease activity. Small colonies occur on agar generally due to lack of the classical fried-egg appearance, but improved medium has increased colony size, and manganous sulfate or calcium chloride, both sensitive indicators of ammonia, result in dark brown Ureaplasma colonies. (Shepard and Masover, 1979; Taylor-Robinson and Gourlay, 1984; Taylor-Robinson, 1989)
The multiple banded antigen (MBA) gene is present in all serovar of Ureaplasma (Teng et al., 1994). This gene appears to play a significant role in the organism's virulence (Kong et al., 1999), and the gene's 5′ regions are markers of biovar specificity and diversity (Teng et al., 1994). This region can not only be used to differentiate U. parvum from U. urealyticum, it indicates that there may be 5 MBA genotypes of the U. urealyticum species: A (serovars 2, 5, 8), B (serovar 10), C (serovars 4, 12, 13), D (serovar 9), E (serovars 7, 11). The MBA gene has been cloned and sequenced. (Zheng et al., 1994) The MBA gene consists of a conserved section encoding both a signal peptide and a membrane anchor, and a variable section encoding a number of uniform repeating units. (Zimmerman et al., 2011) Thus, selection of that portion of the MBA gene that codes for a constant region is an excellent target for vaccine or antibody development, in specific embodiments of the invention. The MBA gene for serotype 6 was selected for initial development of the vaccine of the invention, because it is a frequently isolated clinical serotype. (Vancutsem et al., 2008) The MBA appears significant in attachment of the organism. (Monecke et al., 2003; Torres-Morquecho et al., 2010) MBA also appears to activate NF-kappaB through TLR1, TLR2 and TLR6 and induce tumour necrosis factor-alpha (TNFalpha). (Shimizu et al., 2008) The number of MBA variants in vivo is inversely related to the development of clinical inflammation. (Knox et al., 2010)
Simple and rapid methods of Ureaplasma identification have been developed, but now only confirm culture. A solid phase enzyme immunoassay is not reliable. (Taylor-Ronbinson, 1989) A whole chromosome DNA probe was insensitive (especially <103 ccu/ml) and was positive for culture-negative specimens. (Roberts et al., 1987) A PCR for Ureaplasma appears a very good indicator of infection. (Blanchard and Gautier, 1990; Willoughby et al., 1990) and clinical evaluations have confirmed this (Abele-Horn et al., 1996; Blanchard et al., 1993; Cunliffe et al., 1996), but commercial kits are not yet readily available.
Clinical Significance of Organism: Ureaplasma is a sexually transmitted infection associated with a broad range of clinical diseases in men and women including non-gonococcal urethritis, urinary stone formation, suppurative arthritis, and infertility. In men, it causes non-gonococcal urethritis and prostatitis. In women it causes pelvic inflammatory disease, recurrent abortion, chorioamnionitis, stillbirths, premature birth, low birth weight, and postpartum endometritis. In newborn babies it is associated with several diseases including pneumonia, sepsis, meningitis, osteomyelitis, death, intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis (Pediatr. Res. 2011 May; 69(5 Pt 1):442-7, and chronic lung disease. (O'Leary, 1990; Pinna et al., 2006; Waites et al., 2005) However, there is variable occurrence of these diseases in patients colonized with this organism. (Krause and Taylor-Robinson, 1992) The variable development of disease in colonized patients indicates a virulence factor among pathogenic strains, or antibody, variability or both.
Data on the genital tract colonization of non-pregnant women are limited, but appear high in sex workers (44%), STD clinic clients (40%) (Kong et al., 1999), family planning clinic (43%) (Domingues et al., 2002), symptomatic (48%) and asymptomatic (22%) STD clinic patients (Gupta et al., 2008), In addition Ureaplasma has been isolated from the semen of 12% (9% urealyticum, 3% parvum) of all men with infertility compared to 3% (2% parvum, 1% urealyticum) of those who are fertile (Zeighami et al., 2009).
Colonization of the lower genital tract with Ureaplasma in pregnant women is very common varying from 44 to 88%. (Carey et al., 1991; Cassell et al., 1993; Eschenbach, 1993; Kundsin et al., 1996; Luton et al., 1994) Colonization of the lower genital tract with serotype 3 or 6 Ureaplasma is associated with an MBA antibody response to the variable region of these Ureaplasma serotypes in 51% of women while 15% of women who were not-colonized with these organisms demonstrated the same antibody. (Vancutsem et al., 2008)
Colonization of the upper genital tract or amniotic fluid with Ureaplasma in pregnant women appears to be strongly associated with adverse pregnancy outcomes including spontaneous miscarriage, pre-term labor, pre-labor rupture of membranes, and post-partum endometritis and may occur without microscopic or clinical signs of inflammation. (Andrews et al., 1995; Cassell et al., 1983; Font et al., 1995; Gray et al., 1992; Hazan et al., 1995; Horowitz et al., 1995; Kundsin et al., 1996)
The inventors recently completed a prospective case-control study to determine if Ureaplasma colonization or infection of the placenta is associated with an increase in adverse pregnancy outcome, in particular premature birth. (Okunola et al., 2006; Okunola et al., 2007) Two hundred fifty-two women who gave birth at three Baylor affiliated hospitals (St Luke's Episcopal Hospital, Methodist Hospital, and Ben Taub General Hospital) during an 18 month period participated. These women were composed of 3 groups: 58 gave birth to premature infants between 20 and 30 wks gestation; 27 developed perinatal complications (prolonged rupture of membranes >18 hours, premature rupture of membranes, maternal fever >100.4° F., or clinical chorioamnionitis or endometritis) and gave birth to term infants; 167 had no perinatal complications and gave birth to term infants. Over 40% of those women who gave birth to premature infants (p<0.0001) or who had perinatal complications with a term birth (p<0.004), had placental colonization or infection with Ureaplasma, compared to term births without perinatal complication who had a <15% Ureaplasma placental colonization or infection. No maternal demographic, medical, surgical, or pregnancy factors appear to predict Ureaplasma infection or colonization of the placenta. Of the 58 preterm infants, (Molina et al., 2010) 23 placentas were culture positive for Ureaplasma (40%). Infants whose placenta were positive were not different then those who were negative, in either gestation (26±2.4 vs 26±2.1 wks), birth weight (884±278 vs 890±401), male sex (44% vs 54%), race (38% vs 31%), and prenatal factors. 70% of the Ureaplasma were biovar 1, and of those all were either serotypes 3, 6, or 14. Of infants who survived to 36 wks corrected gestational age (CGA), BPD developed in 69% with Ureaplasma in their placenta compared to 37% of those with a negative culture (p=0.062). Of all infants, death or BPD resulted by 36 wks CGA in 78% with Ureaplasma in their placenta compared to 51% of those with a negative culture (p=0.054). Antenatal exposure of the fetus to Ureaplasma may increase the risk of BPD or death. Strategies to prevent Ureaplasma placenta colonization may decrease premature birth and its complications.
To determine those women at risk for placenta colonization, the inventors recently completed a prospective study (Weisman et al., 2009) of 290 women evaluating Ureaplasma vaginal colonization, and the following was observed: 44% of women at 16 wks gestation had vaginal Ureaplasma colonization; colonization did not change significantly throughout gestation; 32% of all colonized women developed placental Ureaplasma infection (12% of all); all women with placental Ureaplasma infection had vaginal colonization at 16 wks gestation. In preterm births: 67% had vaginal colonization; this did not change throughout gestation; 62% of colonized women developed placental Ureaplasma infection (42% of all). Vaginal colonization at 16 wks gestation is an early marker for those at risk of poor pregnancy outcome and potential target intervention, in certain cases of the invention. Although other conditions (e.g. other infections, anatomic abnormalities, endocrine disorders, maternal medical conditions, etc.) may contribute to poor pregnancy outcome, Ureaplasma colonization of the placenta appears a significant association. If those at risk for poor outcome can be identified early, intervention strategies including antibiotics or more likely vaccines could provide protection from Ureaplasma and adverse pregnancy outcomes.
It has been proposed that Ureaplasma should be eradicated from the urogenital tracts of women and their partners. (Kundsin et al., 1996) Ureaplasma is not susceptible in vitro to penicillins, sulfonamides, trimethoprim, aminoglycosides, and clindamycin, but are generally (about 90%) susceptible in-vitro to tetracyclines, and variably to macrolides (e.g. erythromycin). (Cassell et al., 1993) The inventors have confirmed in recent studies the variable susceptibility of Ureaplasma to erythromycin in vitro. In view of the high colonization rate and sexual transmission rates of Ureaplasma, it is unlikely that such strategies will be effective in its eradication. In addition, this organism has been observed to persist in the genital tract despite antibiotic treatment. In couples attending an infertility clinic this organism persisted in the genital tract despite antibiotic treatment. (Hipp et al., 1983) Routine use of intraoperative prophylactic-antimicrobial therapy at Cesarean delivery did not effect Ureaplasma colonization of the chorioamnion at delivery. (Andrews et al., 1995) Macrolides (Eschenbach et al., 1991; Mazor et al., 1993; Romero et al., 1993) have not been reliable in eradicating genital tract Ureaplasma or adverse perinatal outcomes in two randomized controlled trials. Although newer antibiotics such as glycylcyclines (Kenny and Cartwright, 1994) and quinolones (Kenny and Cartwright, 1996) may prove more effective, their safety and efficacy during pregnancy are unproven.
It has been suggested, but not demonstrated, that lack of specific antibody may be critical for preventing Ureaplasma infection, because specific protein antibody may inhibit growth in vitro. (Cassell et al., 1993) Hypogammaglobulinemic patients have an increased susceptibility to Ureaplasma. (Taylor-Robinson et al., 1986) Serological studies of hypogammaglobulinemic patients (Volger et al., 1985), pre-term infants (Quinn et al., 1983), and women with recurrent spontaneous abortions (Quinn et al., 1983) support this concept. Increased susceptibility of infants of <30 wks gestational age to Ureaplasma induced respiratory disease may be related to their hypogammaglobulinemia (Ballow et al., 1986) or to their lack of specific antibody (Cassell et al., 1988; Cassell et al., 1988).
It has been suggested, but not demonstrated, that monoclonal antibodies to specific protein antigens of Ureaplasma can inhibit growth of these organisms in vitro and indicates that specific antibody may be important for host defense. (Watson et al., 1990) There is a long-felt need in the art to provide useful methods and reagents for Ureaplasma vaccines and methods and compositions to prevent or treat Ureaplasma infection.